Molecular imaging to quantify tumor receptor activity has incredible potential for imaging extracellular protein signaling, yet many ligand molecules cannot be used effectively, because they do not differentiate between simple vascular permeability based uptake and true receptor binding. The development of methodologies which allow quantitative uptake of molecules in tumors is a primary focus of this grant in the renewal period. Fluorescence imaging of receptor activity in vivo is done using a novel Magnetic Resonance-guided Optical (MRgO) imaging instrument that is customized for small animals, with a primary focus on glioma tumor studies. The hardware and software sub- systems will be developed further to provide 4 layer volumetric data of fluorescence tomography guided by the MR data, and overlayed for visualization of the entire orthotopic tumor. The goal of this work is to analyze how molecular tracers of glioma tumors can be used to effectively quantify tumor receptor activity, in addition to vascular perfusion from contrast MRI. The small molecule epidermal growth factor (EGF) is used as a probe in conjunction with a non-specific fluorophore agent to show that ratiometric measurements of the uptake could lead to quantification of the bound EGF Receptor uptake. Glioma tumors that are microinvasive and therefore are not detectable with typical MR imaging can be detected with this new probe molecule, and the dual probe method will be used to show how MRgO imaging can provide a fundamentally new method to image this disease. The design of high and low affinity probes of difference molecular weights for ratiometric imaging is studied in the system with ex vivo validation, to confirm that the in vivo images are truly reflective of the tissue concentration levels. The project ends with the study of microinvasion and testing the detection of this spontaneously and in the setting of anti-vascular therapy. The grant focuses on the technology development, methodology development about how optical fluorescence can be added usefully to imaging science, with these two potential applications in cancer.